Anti-latent TGFβ binding protein 4 antibody improves muscle function and reduces muscle fibrosis in muscular dystrophy

Duchenne muscular dystrophy, like other muscular dystrophies, is a progressive disorder hallmarked by muscle degeneration, inflammation, and fibrosis. Latent transforming growth factor β (TGFβ) binding protein 4 (LTBP4) is an extracellular matrix protein found in muscle. LTBP4 sequesters and inhibits a precursor form of TGFβ. LTBP4 was originally identified from a genome-wide search for genetic modifiers of muscular dystrophy in mice, where there are two different alleles. The protective form of LTBP4, which contains an insertion of 12 amino acids in the protein’s hinge region, was linked to increased sequestration of latent TGFβ, enhanced muscle membrane stability, and reduced muscle fibrosis. The deleterious form of LTBP4 protein, lacking 12 amino acids, was more susceptible to proteolysis and promoted release of latent TGF-β, and together, these data underscored the functional role of LTBP4’s hinge. Here, we generated a monoclonal human anti-LTBP4 antibody directed toward LTBP4’s hinge region. In vitro, anti-LTBP4 bound LTBP4 protein and reduced LTBP4 proteolytic cleavage. In isolated myofibers, the LTBP4 antibody stabilized the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, the anti-LTBP4 in combination with prednisone, a standard of care for Duchenne muscular dystrophy, further enhanced muscle function and protected against injury in mdx mice. These data demonstrate the potential of anti-LTBP4 antibodies to treat muscular dystrophy.

used are as follows: monoclonal anti-LTBP4 at 1:500; rabbit polyclonal anti-Xpress at 1:500. Donkey anti-human and goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (709-035-149 and 111-035-003; Jackson ImmunoResearch) were used at a dilution of 1:2500. SuperSignal™ West Pico Chemiluminescent Substrate and SuperSignal™ West Femto Maximum Sensitivity Substrate (34080 and 34096; Thermo Fisher Scientific) were applied to membranes and membranes were visualized using an Invitrogen™ iBright™ CL1000 Imaging System (A32749; Thermo Fisher Scientific). Pierce™ Reversible Protein Stain Kit for PVDF Membranes (24585; Thermo Fisher Scientific) was used to stain the blot to ensure complete transfer and equal loading. Immunoblot bands were quantified using FIJI gel analysis tools.

Injury Analyses
Injury area 3hrs after injection. Mouse TA muscle was cross-sectioned. Four sections per muscle were saved every 30µm, spanning 120µm of muscle. Sections were fixed for 5 mins in 4% paraformaldehyde, were rinsed in PBS, and then stained with WGA-488 used at 5µg/ml (W11261;Thermo Fisher Scientific) and Hoechst 33342 used at 1:10,000 (H3570; Thermo Fisher Scientific). Slides were mounted in ProLong® Gold Antifade Mountant. The entire muscle section was imaged using the stitching feature on a Keyonce microscope using a 4x objective. All sections were imaged identically using the same exposures. Injury area ( calculated as (EBD-positive area / by total muscle area) *100)) and Evan's blue dye uptake fluorescence was measured as integrated density was measured using FIJI (NIH). Surface plot profiles were generated in FIJI (NIH). Sixteen of 20 samples passed quality control and were included in the analysis. Excluded samples were as follows: one sample was excluded from analysis due to an incomplete cardiotoxin injection, two samples were excluded due to containing only a partial muscle section, and one sample was excluded that required additional processing / handling steps and subsequently was >2 standard deviations above the mean outside the 95% CI.
Analyses done on muscle seven days after injury. Mouse TA muscle was sectioned in crosssection and stained with hematoxylin and eosin (H & E). The entire muscle section was then imaged with a Keyonce microscope at 10x with the tiling feature. The region of injury was measured using FIJI (NIH) and expressed as a percentage of the total cross-sectional area of the muscle measured with FIJI(50). Internal nuclei were counted in the region of cardiotoxininduced injury from laminin/Hoechst immunostained sections. Internal nuclei were counted per myofiber using FIJI (NIH). Data was expressed as % of myofibers containing x number of nuclei with >200 myofibers per mouse counted and >1000 myofibers analyzed per treatment group. Myofiber cross-sectional area was measured in the region of cardiotoxin-induced injury from laminin immunostained sections using SMASH. >350 myofibers per mouse were measured and >2500 myofibers analyzed per treatment group.
Physiologic analyses. Mice were weighed once per week over the course of 4 weeks (shortterm experiments) or 24 weeks (long-term experiments). Weekly body weight informed the injected dosage volume. For long-term treated mice, grip strength, serum collection, echocardiography, and ECG were performed at study start and then once every 8 weeks for 24 weeks. Analyses were done blinded to genotype and treatment.
Body weight and muscle analysis. Mice were weighed weekly over 24 weeks. At sacrifice tibia lengths were measured, and raw body mass was normalized to the average tibia length. Muscles were removed, immediately weighed and normalized to tibia length. The midportion of the quadriceps, triceps, gastrocnemius/soleus, and gluteus/hamstrings muscle was used for histological analysis, while the remaining portion was minced and used for EBD and HOP quantitation. Excised muscles were immediately frozen in liquid nitrogen, placed in pre-cooled Nalgene cryovials and stored at -80 o C or placed in Fisher HealthCare™ PROTOCOL™10% buffered formalin (23-305510; Thermo Fisher Scientific).
Grip strength. Grip strength assessments were performed at day 0 and then once every 8 weeks using a Chantillon Ametek Force Transducer in a Columbus Instruments apparatus as described (47). All assessments were performed by the same operator performed who was blinded to the animal's genotype.
Serum collection. Blood was collected by means of retro-orbital puncture with heparinized capillary tubes (20-362-566; Thermo Fisher Scientific) into Microtainer™ Gold Top Serum Separator (365967 Becton Dickinson) and centrifuged at 8,000 x g for 10 minutes. The plasma fractions were frozen and stored at −80°C.
Serum biomarkers. Serum creatine kinase (CK) was analyzed in triplicate for each mouse using the EnzyChrom Creatine Kinase Assay Kit (ECPK-100; BioAssay Systems) following manufacturer's instructions. Results were acquired with the Synergy HTX multi-mode plate reader (BioTek®).
Electrocardiogram. Surface electrocardiogram recordings and analysis were performed on conscious mice using the ECGenie apparatus (Mouse Specifics). Mice were allowed to acclimatize to the ECGenie platform for five minutes and then data was collected using LabChart (ADInstruments). Four segments of 2-4 second long runs of clean ECG signal were analyzed using the ECGenie software and averaged into single data points per animal.
In-situ force and fatigue. Muscle mechanics were performed as described previously (47, 51) on the tibialis anterior muscle using a Whole Mouse Test System (Cat #1300A; Aurora Scientific) with a 1N dual-action lever arm force transducer (300C-LR, Aurora Scientific). During study, animals were anesthetized (0.8 l/min of 1.5% isoflurane in 100% O 2 ). Both muscles per animal were assayed. Tetanic isometric contraction was induced as follows: initial delay, 0.1 sec; frequency, 200Hz; pulse width, 0.5 msec; duration, 0.5 sec; using 100mA stimulation. Length was adjusted to a fixed baseline of 50mN resting tension. Specific force equaled tetanic force normalized to muscle cross-sectional area for each muscle. Fatigue analysis used repeated tetanic contractions every 10 seconds for 25 cycles (complete exhaustion).

Eccentric contraction (ECC) induced injury.
Eccentric contraction-based muscle injury was performed on tibialis anterior muscles of anesthetized mice (0.8 l/min of 1.5% isoflurane in 100% O 2 ) using a Whole Mouse Test System (1300A; Aurora Scientific) with a 1N dual-action lever arm force transducer (300C-LR, Aurora Scientific). Both muscles per animal were assayed. Eccentric contraction train was preceded and followed by two tetanic isometric contractions (initial delay, 0.1sec; frequency, 120Hz; pulse width, 0.5msec; duration, 0.5 sec; using 100mA stimulation), each executed 300 sec before last contraction. The eccentric contraction train consisted of four eccentric stimuli imparted on muscle every 100 sec using the following specifications: tetanus (100mA, 250hz, 0.1msec pulse width, 120ms duration); during tetanus, 20msec-long inward lever flexion (-5 in channel units) followed by 100msec-long outward lever flexion (+5 in channel units). Muscle tension baseline was adjusted to 50mN before the initial tetani, before the eccentric contraction train and again before final tetani. Force loss was calculated as mean initial recorded maximum tetanic force (derived from the two initial tetani) minus mean final recorded maximum tetanic force (derived from the two final tetani). Specific force was calculated as tetanic force normalized to muscle cross-sectional area assayed for each muscle from every animal.
Evans Blue dye uptake quantification. Mice were injected IP with 5µl/g of 10µM EBD (E2129; Sigma-Aldrich). Mice were sacrificed ~24hrs after injection. Multiple muscle groups were assessed including the abdominal, diaphragm, quadriceps, gastrocnemius/soleus, gluteus/hamstrings, and triceps and values normalized to tissue weight and kidney dye uptake. Each sample was assessed in duplicate. Absorbance was measured at 620 nm on a Synergy HTX multi-mode plate reader (BioTek®). Results are reported as arbitrary OD units/mg of tissue.

Muscle analysis.
Mouse TA muscles from long-term treated mice were dissected and frozen in liquid nitrogen. Anti-laminin or anti-caveolin sarcolemmal fluorescence outlined individual myofibers and was used to assess the myofiber mean cross-sectional area (CSA) automatically using FIJI (NIH). n>11 mice per treatment from at least five fields per mouse. The percentage of fibers with central nuclei was calculated from the number of fibers containing internalized nuclei in each image/the total number of fibers counted per image, standardized as a percentage. At least 500 fibers per treatment were analyzed (n>5 from each genotype). Images were captured using a Zeiss Axiophot microscope.
Quantitative RT-PCR. Gene expression analysis was conducted on total RNA extracted with TRIzol (catalog 15596018, Life Technologies) from gastrocnemius and spleen tissues per manufacturer's instructions. RNA was isolated from approximately 50 mg tissue and 2g RNA was used per reverse transcription reaction. Each cDNA reaction was obtained using qSCRIPT cDNA supermix assay in 20l reactions per manufacturer's instructions (catalog 95048, QuantaBio). cDNA was diluted 1:10, and 2μl was used per 10 μl qPCR reaction. Each 10 μl qPCR reaction contained 100 nM primers and 5μl iTaq SYBR Green Mix (catalog 1725124, Bio-Rad). Primers to gzmb: GZMB Fw-ACAAAGGCAGGGGAGATCAT and GZMB Rev-CGAATAAGGAAGCCCCCACA. RN45S was used as a control (RN45S Fw: gtaacccgttgaacccgatt, RN45S Rev: ccatccaatcggtagtagcg). SybrGreen Fluorescence was quantitated using the CFX96 Real-Time System (Bio-Rad; thermal profile: 95C 3min, 40 cycles of 95C 15sec, 60C 45sec) and gene expression values were analyzed as fold change to control tissues. Figure S1. Mouse anti-LTBP4 monoclonal SBI-3m antibody recognizes human and mouse LTBP4. LTBP4 migrates at ~160 kDa on gel electrophoresis (green arrow). A) Mouse anti-LTBP4 mononclonal antibody generation through backbone engineering. B) Mouse anti-LTBP4 antibody SBI-3m recognized recombinant human LTBP4 protein by immunoblot. C) SBI-3m recognized overexpressed mouse LTBP4 protein, including both the 129 and D2 forms, after overexpression in HEK cell lysates. UT = untransfected HEK lysates. Figure S2. Human anti-LTBP4 monoclonal antibody was detectable at muscle membranes for at least 21 days after a single antibody injection. mdx/hLTBP4 male mice were injected i.p. with SBI-3h at 10 mg/kg once and sacrificed 1, 7, 14, or 21 days later. Control mice were injected with PBS once and sacrificed 1 day later. Muscles were harvested and probed with anti-human IgG secondary antibodies. Longitudinal imaging of the extensor digitorum longus (EDL) muscle detected the presence of costameric, anti-human IgG fluorescence signal (green) as much as 21 days after injection of human anti-LTBP4 antibody (SBI-3h), consistent with target engagement. Costameric fluorescence was not detected in PBS-injected control mice. Scale bar 10 µm. Figure S3. Anti-LTBP4 protects muscle from injury in vivo. mdx mice were pretreated with 10 mg/kg anti-LTBP4 antibody or PBS every 4 days for 2 weeks. Mice were then injected with Evan's blue dye (EBD). Two hours after dye injection, the tibialis anterior muscle was injured with cardiotoxin. Muscle was allowed to recover for 3 hours after cardiotoxin injury to capture an early phase of injury response to assess whether LTBP4 antibody protects against injury. Anti-LTBP4 treatment reduced EBD-positive (red) injury area and reduced EBD fluorescence measured as integrated density compared to controls. Representative images and 3-D surface plot profiles are shown. WGA conjugated to 488 (green) was used to outline the muscle. n  8 legs per group. *P<0.05 by Mann-Whitney test. Figure S4. Cardiac outcomes after long-term SBI-3h antibody treatment. mdx/hLTBP4 male mice were injected i.p. with either PBS, human IgG control, SBI-3m or SBI-3h at 10mg/kg once per week for 24-weeks. A) Cardiac fractional shortening (FS%) was increased in mice treated with SBI-3m compared to PBS controls. B-C) No significant changes in PR or QRS intervals were noted between any treatment groups. * P<0.05 by one-way ANOVA. (panel A & B, n = 11; panels C & D n  4) mice per group.